Beginning a century ago with the Golgi stain technique, neuronal labeling has played a central role in the study of anatomical and physiological systems. The Golgi stain technique utilizes silver impregnation of fixed tissues and allows imaging of the smallest objects that are resolvable using a light microscope. Using the Golgi technique, entire neurons, including the dendrites and axons, could be imaged for the first time. Subsequently, fluorescence techniques were developed in order to study living neural preparations. Fluorescent dyes that vitally label organelles concentrated in synapses (Magrassi et al., J Neurosci., 1987, 7:1207-14) or that mark synapses due to vesicular recycling (Lichtman et al., 1985, Nature 314:357-9; Betz et al., J. Neurosci., 1992, 12:363-75) have provided a means to study dynamic aspects of neuronal connections.
Recently, dipyrrometheneboron difluoride labeled flourescent microparticles used for labeling were disclosed in Haugland et al., U.S. Pat. No. 5,723,218. Haugland et al. teach the use of derivatives of dipyrrometheneboron difluoride dyes incorporated internally into polymeric microparticles having a diameter of between 0.01 micrometers and about 50 micrometers in order to produce a dye impregnated particle that can fluoresce for extended periods of time. Thus, in Haugland et al., the dye is not released from the particle, but the particles are themselves used as fluorescent indicators.
A method of labeling cells using a series of fluorescent dyes was also developed by Singer et al., U.S. Pat. No. 5,573,909. Singer et al. disclose methods for labeling target materials using a series of particles that internally incorporate two or more dyes having overlapping excitation and emission spectra. Both a donor and a transfer dye are used. According to Singer et al., the target materials are labeled by combining the particles with a sample containing the target material. The Singer technique requires the use of a two-step process in which the sample to be studied is first pre-labeled with a fluorescent marker such as an antibody. Subsequently, the particles are added to the sample such that the fluorescent antibody interacts with the particles to generate fluorescence resonance energy transfer (FRET), which is detected with a camera. The dye is not released from the particles in the Singer method. Therefore, the entire surface of a target cell is not labeled.
One approach which has been developed to label an entire cell is the introduction and expression of Green Fluorescent Protein (GFP). GFP and its variants have been used to label cells in transgenic animals (e.g., Chalfie et al., 1994, Science, 263:802-805; van den Pol and Ghosh, 1998, J. Neurosci., 18:10640-10651). GFP is a spontaneously fluorescent protein isolated from coelenterates, such as the Pacific jellyfish, Aequoria victoria. GFP serves to transduce, by energy transfer, the blue chemiluminescence of another protein, aequirin, into green fluorescent light. GFP can also function as a protein tag, as it tolerates N— and C— terminal fusion to a broad variety of proteins, many of which have been shown to retain native function. When expressed in mammalian cells, fluorescence from wild type GFP is typically distributed throughout the cytoplasm and nucleus, but excluded from the nucleolus and vesicular organelles. The enormous flexibility of this material as a noninvasive marker in living cells allows for its use in numerous other applications such as a cell lineage tracer, reporter of gene expression, and as a potential measure of protein—protein interactions. Particle mediated gene transfer and viral transfection techniques (Lo et al., 1994, Neuron 13:1263-1268; Vasquez et al., 1998, Exp. Neurol., 154:353-365.) have been successfully used to label cells with GFP. These newer techniques provide the kind of resolution obtained with the Golgi technique, but can be used in vivo.
Current vital labeling techniques have several limitations. First, it is difficult to label individual cells, such as neurons, differentially within complex biological networks. Although lipophilic carbocyanine dyes are available at many different excitation and emission profiles, they are typically applied to cells and tissues in ways that label many individual cells at the same wavelength (e.g., O'Rourke et al., 1994, Neuron, 12:921-934; Wu and Cline, 1998, Science, 279:222-226.). For example, according to one known technique, lipophilic crystals are placed on nerves or clusters of nerves causing tens or hundreds of neighboring cells to be labeled the same color (See Nakamura and O'Leary, 1989, J. Neurosci., 9:3776-3795; Nakamoto et al. 1996, Cell, 86: 755-766). Pressure injection of carbocyanic dyes into cells is another technique that has been used (See, O'Rourke et al., 1994, Neuron, 12: 921-934 (labeling of axonal arbors); O'Rourke et al., 1997, Development, 124: 997-1005 (labeling of migrating cortical cells)). While both of these approaches use carbocyanine dyes, they both have the disadvantage of labeling large patches of cells with a single dye at the site of the application. Therefore, individual cells cannot be differentiated.
Individual cells have been labeled with carbocyanine dyes using iontophoretic injection (Wu and Cline, 1998, Science, 279:222-226). Lipophilic carbocyanine dyes have also been applied with sharp electrodes to label individual axons. (Gan and Macagno, 1995 J. Neurosci. 5:3254-3262; Gan and Lichtman, 1998, Science, 282:1508-1511, Gan et al., 1999, J Neurosci. Methods, 93:13-20.) However, these approaches are tedious, time consuming and only a small number of cells can be labeled at any one time. Attempts have been made to circumvent this limitation by sprinkling the dye crystals onto cells. However, this approach is highly variable because dye crystal size, density and penetration are difficult to control.
GFP expression utilizing gene guns and the use of gene guns for introducing materials into the interior of cells has been described in the following U.S. Patents: Drake, Jr. et al., U.S. Pat No. 4,326,524, Sanford U.S. Pat. No. 5,204,253, Sanford et al. U.S. Pat. No. 5,179,022, Sanford et al. U.S. Pat. No. 5,100,792, Sanford et al. U.S. Pat. No. 5,478,744, Sanford et al. U.S. Pat. No. 5,371,015, Sanford et al. U.S. Pat. No. 5,036,006 and Sanford et al. U.S. Pat. No. 4,945,050. When particles containing DNA encoding GFP enter a nucleus they can result in gene expression (Lo et al. 1994, Neuron, 13:1263-1268). GFP gene expression, however, usually takes more than 6 hours to occur after gene transfection in cell culture and brain slices. This waiting period limits many structural and functional studies, as frequently, considerable structural changes occur, e.g. in brain slices, during the first few hours after preparation (Kirov et al., 1999, J. Neurosci. 19:2876-86). In addition, the variety of distinct emission profiles available using this approach is limited. In GFP labeling, the emission profiles are directly related to the structure of the native protein which emits fluorescence. Currently, cells labeled using GFP labeling techniques may only be imaged at a relatively few non-overlapping excitation peaks. Further, the GFP transfection technique is very inefficient despite the use of high density particle delivery to the desired tissue with only a small fraction of cells penetrated by the particles expressing GFP. This limitation prevents the use of GFP transfection for the study of networks of neurons, because such studies require high density labeling such that the interactions between adjacent cells can be optically imaged.
A critical need exists, therefore, for a method of rapid, individualized labeling in cell culture or tissues that allows adjacent cells to be optically separated despite the use of high density labeling in both fixed and living tissues. As described below, Applicants have discovered such a method of rapid and individual labeling using microparticles that meets this need.